Fig. 2: Validation of HDACis as EVI1 modulators.
From: Orthogonal proteogenomic analysis identifies the druggable PA2G4-MYC axis in 3q26 AML
A EVI1 and ∆EVI1 expression in EVI1High AML cell lines (n = 3 biological replicates). B Nuclear localization expression of EVI1 (in red) detected by immunofluorescence (IF) in EVI1High AML cell lines (n = 3 biological replicates). Cell nuclei were stained with DAPI (blue), scale bar: 10 µm. C EVI1 and ∆EVI1 expression in TF1 cells 6 days after shRNA transduction. D Percentage of EVI1 mRNA relative to the control gene RPL13A (ΔΔCT) in TF1 cells 6 days after shRNA transduction. E Effect of EVI1 loss in TF1 cells at three or six days after shRNA selection on cell proliferation. F Effect of shRNA-directed EVI1 loss in the ability of TF1 to form colonies in methylcellulose compared to NT (n = 2 biological replicates). Scale bar: 1000 µm. The histogram on the right shows the mean number of colonies per field 20 days after plating cells. NT = non-targeting, sh#16 and sh#87 = shRNA directed against EVI1 (C–F). G Effect of AR-42, belinostat, and entinostat on cell viability in EVI1High cell lines following 72 hours (hr) of treatment. H Annexin V/propidium iodide (PI) staining after 72 hr of HDACis treatment. Events ≥ 20,000. I Apoptotic fold increase, expressed as a percentage of annexin V-positive cells relative to vehicle control. J Effect of HDACis on the ability of 3q26 AML cell lines to form colonies in methylcellulose. Representative images of UCSD/AML1 treated with vehicle (DMSO), 0.5 µM AR-42, 0.5 µM belinostat, and 0.5 µM entinostat (n = 3 biological replicates). Scale bar: 1000 µm. K The histogram shows the mean number of colonies per field 20 days after plating cells (n = 3 biological replicates). Statistical significance among groups was determined by one-way (D, K) or two-way (E) ANOVA using Tukey’s correction for multiple comparison testing. Data are presented as mean ± standard deviation (SD) in D (n = 3), E (n = 4), F (n = 10 fields per condition), G (n = 2), and K (n = 10 fields per condition). Source data are provided as a Source Data file. See also Supplementary Fig. 3.
